Background We are testing several approaches, including gene therapy, small molecule screening for drug discovery, and retinal reconstruction. All strategies are guided by the knowledge of molecular mechanisms of photoreceptor development, homeostasis, and disease acquired through other projects (see EY000450-73-75). Gene therapy In collaboration with P. Colosi and T. Li, we initiated pre-clinical studies for the treatment of Leber congenital amaurosis (LCA) caused by CEP290 mutations and of retinitis pigmentosa (RP) caused by mutations in the RPGR and RP2 genes. We focused on animal models and genetic characterizations, whereas P. Colosi's unit was involved in AAV-based gene therapy. With the departure of Dr. Colosi, his former staff scientist Dr. Zhijian Wu will be leading the gene therapy efforts and collaborate extensively with our section. Our collaboration with S. Jacobson, G. Aguirre and W. Hauswirth has demonstrated the efficacy of gene augmentation therapy in two blinding canine photoreceptor disease models for RPGR-XLRP. We continue to collaborate with Dr. Jacobson to develop the path for human treatment. Drug discovery We are developing reagents and protocols for high throughput screens (HTS) of small molecules with a goal to discover new drugs to prevent photoreceptor degeneration. We are using zebrafish as animal model, developing in vitro cell-based assays, and deriving photoreceptors from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) for small molecule screening. . CRX and NRL are two transcription factors essential for photoreceptor homeostasis and, when mutated, cause photoreceptor dysfunction and death. We have used TALENS to generate mutations in the zebrafish homologues of both. We have obtained several founders and are using F1 carriers to generate homozygous lines and select them based on their degeneration phenotype. Crossing the transgenic lines with the reporter lines TalphaCP-GFP and Rho-GFP lines, in which GFP expression is restricted to cones or rods, respectively, will provide the models needed for our screening purposes. We have immortalized a fibroblast cell line derived from Cc2d2a-/- mouse embryos (see EY000450) and stably expressed a reporter gene to evaluate cilia formation. Loss of Cc2d2a in mice (a model of Meckel syndrome) leads to embryonic lethality and pleiotropic phenotypes in the absence of cilia. We will test small molecules that induce cilia biogenesis in the fibroblast cell line. Such molecules will help us elucidate pathways of cilia biogenesis and function that are affected in ciliopathies. By HTS we aim to identify small molecules that facilitate the generation of rod and cone photoreceptors using zebrafish, hESC and hiPSC cultures. Upon screening the NCI Diversity II set (2000 compounds) and The Spectrum Collections (3000 compounds) chemical libraries using two zebrafish transgenic reporter lines (Rho-GFP and TalphaCP-GFP), we have identified eight potential compounds that decrease the reporter signal in rods or cone and one that increases the reporter signal in cones. We are validating these results. As the identity and function of these compounds is established, these studies will help us understand pathways underlying photoreceptor development and homeostasis. To identify small molecules that direct and enhance differentiation of hESCCs into rod or cone photoreceptors, we are testing experimental conditions using hESC (H9) expressing green fluorescent protein (GFP) under control of Crx promoter, in which GFP is turned on in post-mitotic photoreceptor precursors. The protocol entails induction of hESC to early neural lineage after embryoid body (EB) formation. Upon detection of GFP, EBs are dissociated into single cells and plated for screening. Green fluorescence is detected and used for quantification. We have established the optimal cell number for 96 and 384 well plates and an optimal range of the GFP signal for the image detection system. We are now trying to scale up the assay to 1536 well plate in preparation for the first pilot small molecule screening. We are testing known neuroprotective factors as an alternative approach to preserve photoreceptors by blocking cell death and/or strengthening endogenous pro-survival pathways. We focused on CNTF, one of the most efficient neurotrophic factor studied in the retina, which is currently being tested in several clinical trials. We aim to identify pathways or molecules downstream of the CNTF signal that mediate its neuroprotective effects in the retina and in rod photoreceptors. By analyzing RNAseq data of CNTF-treated retina in a time-course we obtained preliminary indications of the involvement of immune response (interferon) signaling and energy production in mitochondria. Use of iPSCs and ESCs to develop therapies Replacement of dysfunctional and/or dying photoreceptors offers a promising approach for the treatment of retinal degenerative diseases at late stages, when the paucity of photoreceptors negates gene and pharmacological interventions. We previously showed that Nrl-expressing newborn rod photoreceptors can differentiate and integrate in degenerating mouse retina, thus suggesting the feasibility of retinal repair by photoreceptor replacement. Since then, others and we have been pursuing avenues to increase the efficiency of integration. We have successfully transplanted rod photoreceptors precursors dissociated from postnatal Nrlp-eGFP mice into Nrl-/-:Cep290rd16 mice, a model of LCA that reflects the residual-cone scenario in LCA patients and that we have extensively characterized. The transplanted cells formed outer segments with polarization toward the retinal pigment epithelium and developed synapses, interacting with host bipolar cells. Scotopic a-wave was partially reversed by transplantation. Matrix-remodeling adjuvants increased integration efficiencies. In collaboration with M. Takahashi at the Riken Center for Developmental Biology, we evaluated Ca(2+) responses and cell membrane currents of rod precursors from Nrlp-eGFP mice and derived from iPS cells established from Nrlp-eGFP mice, before and after transplantation in the subretinal space. We showed that GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2+) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures after transplantation into the retina of Crx-/- mice and show functional maturation. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar physiological responses as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We have established parameters that define photoreceptor function, showed that photoreceptors differentiated from transplanted developing rods display comparable currents to native rod photoreceptors and that iPS cell-derived rods have properties similar to developing rods (1). We have successfully differentiated into retinal neurons human iPSCs and ESCs stably expressing fluorescence reporter construct and we are testing lineage-restricted cells (as determined by the expression of specific promoter constructs) for transplantation in the degenerating mouse retina. Functional characterization of stem cell-derived photoreceptors in vitro and after transplantation in the recipient mouse is conducted using calcium imaging and patch clamp recording. Based on RNA-Seq analysis of lineage-restricted cells, we will identify cell surface markers that will allow us to purify sub-populations of differentiating cells for transplantation and to establish which one has the best therapeutic effect.